1. Field of the Invention
A mobile equipment system for gene diagnostics.
2. Description of the Related Art
The examination of diagnostically relevant biological samples, such as serum, plasma, blood, swab samples, or organ smears, for the detection of infectious pathogens has gained tremendous importance in recent years. Viral infections such as HIV, HCV, or HBV are increasing worldwide. Furthermore, bacterial infections are also increasing again, among other things also as the result of climatic changes. The occurrence of new, deadly infectious diseases having an extremely high infection potential (SARS, bird flu) shows more and more clearly that rapid diagnostics, which can be carried out on site, are decisive for preventing epidemics. Furthermore, diagnostic systems that are easy to handle and relatively inexpensive also play an important role, particularly for developing countries, in combating the spread of infectious diseases. The majority of the tests currently being used primarily for the detection of viral infectious diseases (HIV, HCV, HBV) are based on carrying out REAL-TIME PCRs. These tests are tied to extremely expensive equipment technology prerequisites, and also expensive reagents. These methods can only be carried out by trained technical personnel in special laboratories. On-site diagnostics are not possible. New generations of integrative system solutions (combinations of nucleic acid isolation, amplification, and chip detection) for mobile on-site diagnostics are in development, but not in a stage of successful marketing. Furthermore, these systems often focus on the sector of military diagnostics, and, in analogy to the “traditional REAL-TIME PCR” methods, are also very expensive, in terms of both devices and reagents.
The so-called rapid PCR technology allows carrying out amplification reactions within only a few minutes and is therefore clearly faster than standard PCR methods. For the miniaturized device solution, the invention is accomplished by means of a novel reaction cavity (consumable). In the sector of PCR consumables, various methods are known for their production. The most widespread is the production method using injection-molding. The consumables produced in this manner are commercially available in a large number of shapes and arrangements. However, these receptacles all have a very thick wall thickness (approximately 0.2 mm-0.35 mm), which opposes good heat transfer from the heated sample block into the sample, as a very great resistance. If these receptacles are used as intended, in a thermocycler, the sample block situated in the devices heats up at a speed of up to 5° C./s. The resulting speed in the sample, however, is significantly reduced by the receptacles, so that the sample (situated in the receptacle) is only heated up at approximately 1.5-2° C./s. Even those receptacles offered for sale as “thin-wall” receptacles (minimal wall thickness of approximately 0.19 mm; Eppendorf product catalog 07-08, p. 200) are not able to significantly improve this condition. The technical prerequisites (heating and cooling) for rapid processing of the temperatures in PCR are implemented technically, but the efficacy is greatly impaired, because of the consumable used.
Usual sample volumes used in PCR lie in the range of 5-50 μl. The receptacles, produced by means of injection-molding, possess their maximal wall thickness at the bottom, so that tempering and implementation of a PCR reaction of low volumes, 1-10 μl (which collect at the bottom of the receptacles), can be carried out only very imprecisely and slowly.
The metallic sample blocks used (aluminum, silver) in thermocyclers having a very great heat capacity furthermore ensure that rapid temperature changes in the sample are impossible to implement, even when using strong heating elements. The rapidity of existing systems on the market, which work on the basis of metallic sample blocks and injection-molded consumables, thus comes up against objective limits. From this, it follows that because of their tremendous thermal inefficiency, miniaturized, mobile variants of these thermocyclers exceed the electrical conductivity of accessible battery technology in unacceptable manner.
Other stationary systems bet on advanced technology to increase the thermal efficiency and make rapid PCR possible.
The most well-known commercially available PCR system that will be mentioned here is the LightCycler from Roche Molecular Biochemicals (cat. No. 1909339 and cat. No. 2011468). This system is based on the use of very thin glass capillaries as PCR consumables and carries out tempering by means of hot air that flows around the capillaries. These capillaries can hold a volume of 10-20 μl and are characterized by their large surface area, which allows good heat transfer. However, this large glass surface area absorbs components of standard PCR batches and thus causes the reaction to become more and more inactive. In order to compensate this effect, different carrier molecules, etc., for example, have to be used (EP 1133359). Another disadvantage in this connection is the handling of the very thin capillaries and their price. Miniaturization and simple handling of the receptacles continues to be impossible, however.
A technology that is not based on standard consumables and can be used for mobile use is explained in the published patent application US2005/0227275 A1. Here, a woven metal textile is introduced into the wall of the PCR reaction chamber, by means of production technology. This ensures direct heat transfer to the sample. Although it is pointed out that the wall should be thin, it is not explained how thin. At the same time, cooling of the sample is not discussed in any way. Thus, while this reference explains that rapid PCR is supposed to be carried out, it does not, however, provide sufficient information about technical implementation. This point also includes the fact that the consumable described there does not meet the requirements of being a cost-advantageous consumable material. Introduction of a defined woven heating material and a device for temperature control stand in the way of this requirement. Furthermore, the object according to the invention of the published patent application U.S. 2005/0227275 A1 is furthermore carrying out a PCR reaction and detecting amplification products by means of a lateral-flow strip. PCR with marked primers and nucleotides is described (claim 1, FIGS. 1, 2, 3, 4, 6). However, a disadvantage of this method is that the PCR product is not hybridized with a marked probe. The sole detection of an amplification product in this manner is diagnostically very uncertain, however, since the required 100% specificity of the amplification product is not guaranteed. For this purpose, a specific hybridization reaction is required. Furthermore, there is the latent risk that false-positive results occur due to mis-priming and primer dimers.
The use of a design similar to a “chip” for accommodating a PCR chamber does not represent level of invention, because it is cited many times in the patent literature. Instead, the configuration of the heating/cooling mechanism, of the entire heat transfer, the feed of liquid/solid biochemical reaction components, and the implementation of the process parameter control represent innovation. Thus, for example, the reference WO 2007/092713 A2 discusses a consumable design in chip form that can comprise not only cell sorting and immunological protein detection but also a PCR chamber. Different cell types (precursors of cancer cells, and cancer cells) are separated by means of a lateral-flow test strip method, using appropriate antibodies, and discriminated. However, the lateral-flow method does not serve to detect amplification events. The detection of RNA expression from RNA, which previously took place in a so-called lab-on-a-chip system, is uncoupled from this lab-on-a-chip and takes place as described, on a “laboratory table, using known methods.” It is described that alternatively to this, an amplification reactor and a detector can be integral components of the lab-on-a-chip system. This reference does not discuss the configuration of the PCR chamber in the sense of the aforementioned properties. There is also no mention of a PCR reaction in the claims. Nevertheless, a mobile variant of the equipment system (with consumable) can be discussed here. For operation of the device independent of the power network, power consumption must be optimized and be as low as possible. Because of the use of a large number of thermoelectric modules (“hydrogel ice valves”) and fluid pumps, a person skilled in the art recognizes that this device cannot be suitable for mobile battery operation.
The authors of the reference described above discuss the topic of chip production and of the PCR chamber within this or a similar chip in a publication (Chen, Z., et al.; Ann. N.Y. Acad. Sci. (March 2007) 1098; 429-436). A system is presented that comprises a PCR chamber and a lateral-flow test strip. Measured by the requirements—rapid (rapid PCR), mobile (battery operation), cost-advantageous (consumable), and easy to handle—this invention must be assessed as follows. It is known to an expert that rapid PCR is defined by the total time of the reaction (with 30 cycles in less than 30 minutes) and by the temperature changes in the sample that are achieved (>4 K/second). Without any statement of heating or cooling rates, a cycle time of at least one minute (sum of the stop times without duration of the temperature change), as well as the use of a PCR chamber made of polycarbonate, produced by means of milling technology (wall thickness values of at least 100 μm), it becomes clear to a person skilled in the art that this equipment system cannot be used for rapid PCR. In this publication, a mobile system is not described in any manner. Just like in the patent document described above, more than 10 thermoelectric modules, plus two multidirectional setting valves, a fluid pump, a vacuum pump, and a laser scanner are supposed to be accommodated here, on the equipment side. These modules, which are very power-intensive, as well as the consumable design, which is not optimized, thus preclude even the possibility of mobile battery operation. Finally, a person skilled in the art recognizes that this system cannot be used for mobile use, since the complexity of the consumable (multiple production steps, introduction of hydrogels) requires unreasonable production costs. Detection of the amplification takes place by way of the use of two marked primers. However, it is known to a person skilled in the art that such a detection method on a test strip is highly problematical, since specific amplification products cannot be separated from the non-specific amplificates and so-called primer dimers. Thus, such a detection system cannot be used in diagnostics.
Furthermore, the publication by Wang et al. belongs to the state of the art (Wang, J. et al.; Lab on a Chip (2006) 6: 46-53. The system presented there can be identified by a person skilled in the art as a variant of the two developments described above (for example the same use of ice valves or marked primers as a detection system). Here, the precise properties of the PCR reaction unit are stated once more in concrete terms. A thickness of 250 μm is indicated for the wall between the heating/cooling element and the sample. On the technology side, this chamber and all the channels are introduced into a polycarbonate carrier by means of a CNC milling process. Therefore, the thermal resistance of this chamber still lies above that of commercially available reaction receptacles (wall thickness 200 μm to 300 μm; material polyethylene), in which rapid PCR is only possible by means of high-power equipment. Thus, use of the concept for battery operation is excluded once again. The inefficiency of the concept becomes evident to a person skilled in the art when the authors discuss the difficulties in achieving an acceptable cooling rate. Even with active heat transport by means of Peltier elements and the inclusion of a 14 watt fan (conventional fans for processors require 6 watts), only 2.6 K/second is achieved in the sample (see rapid PCR). The concept must also be critically illuminated with regard to the formulated goal of cost minimization. According to the publication, many different production chains have to be run through in order to produce a chip with this concept. Aside from milling of the precision channels, the surfaces additionally have to be polished, hydrogels have to be introduced, two chip halves have to be joined, using a complicated thermal method, and subsequently, reactivation of the gels has to be implemented. This makes it evident to a person skilled in the art that this chip concept makes a cost-advantageous consumable impossible.